A protocol for rapid, measurable plant tissue culture using stem disc meristem micropropagation of garlic (Allium sativum L.) Gerry Peat and Meriel Jones

ABSTRACT  Plant tissue culture is becoming an important technique for the mass propagation of plants. Problems with existing techniques, such as slow growth and contamination, have restricted the practical work in plant tissue culture carried out in schools. The new protocol using garlic meristematic stem discs explained in this article addresses many of these problems and aims to produce measurable growth over two weeks. These methods could be developed in a range of further investigations.

Plant tissue culture and micropropagation laboratory techniques are becoming economically and ecologically important across the world. In the commercial horticultural world, plant tissue culture techniques enable rapid production of disease-free plants such as Phalaenopsis orchids and Gerbera produced in the Far East, the Netherlands and Madeira, and palm oil palms used in tropical countries to establish biofuelproducing plantations. The benefits of such culture include rapid reproduction, identical flower colour or oil production due to the cloning, and high-density, virus-free plants. In the UK, specialist companies provide contract propagation services to wholesale nurseries, growers and public bodies. From the customer providing a stockplant to the delivery of thousands of rooted shoots takes, on average, 18 months. The techniques are used for conservation purposes, either to grow seeds to maintain genetic diversity, or to bulk up numbers of rare or endangered plants for re-introduction back into the wild. On a Cheadle Hulme School group visit to Micropropagation Services of East Leake, Loughborough, we were shown how cowberry (lingonberry) plants (Vaccinium vitis-idaea) are being propagated using these methods for the reclamation of eroded moorland in the Peak District. The techniques are also being used in industrial and academic research for a range of

purposes, for example to clone varieties and to produce genetically modified plants. The techniques for plant tissue culture were developed in universities in the 1960s and 1970s and at that time it was often used as a research tool, for example to bioassay hormone levels. Methods that could be used in school laboratories were designed and published by Tony Storr (1985), who was working as a teaching fellow for the Standing Conference on Schools’ Science and Technology (SCSST) project presenting industry-related science practicals for schools. He worked in partnership with Unilever plc in Bedfordshire and produced protocols for the culture of cauliflower, carrot callus, Nicotiana and Saintpaulia. From his experience, Storr suggested a success rate of about 80% for these cultures. However, a survey taken at a recent teachers’ conference by the author (GP) shows a different picture, with about 5% uncontaminated measurable growth of cultures. Of the 20 schools represented, 18 indicated that they tried tissue culture on a regular basis because they thought that the techniques were important, but few had any success or expected good uncontaminated growth of cultures. For three decades, this poor success rate has deterred schools from tackling plant tissue culture. All schools in the survey were using versions of the carrot or cauliflower protocol, and all stated that they thought it was SSR June 2012, 93(345)

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Plant tissue culture using stem disc meristem micropropagation of garlic important that students were exposed to the techniques involved in tissue culture.

Problems with existing protocols The methods of culture outlined in the SCSST project and those published elsewhere were trialled in class sessions over the course of two years and practical problems identified. The cultures were set up using aseptic techniques performed on a school laboratory bench rather than using transfer chambers. Medium was produced using recipes given in the SCSST project protocols and sterilised by autoclave. The key problems that were identified are discussed below. Contamination

The major factor in the failure of cultures was contamination by both bacteria and fungi. This resulted in decay and failure of 95% of cultures. The plant material was difficult to sterilise because of surface bacterial and fungal spores in crevices and folds of tissue. Increasing the strength of sterilising fluid or the duration of immersion resulted in damage or death of the plant material. Starting from material grown in greenhouses has been recognised as being more successful because of a reduced bacterial load compared with plants grown in open fields. Fuller and Pizzey (2001) tried to address the problem when culturing cauliflower by adding a biocide called Plant Preservative Mixture™ (PPM™) to the medium at the concentration of 1 cm3 dm−3. Slow growth

Slow growth rates were a major problem because groups that had set up the cultures could only see measurable results after a month or, in many cases, several months. By this time, they had moved on to different topics in class and there was a loss in continuity so they had forgotten the ideas and methods. Insight gained from visits to commercial micropropagation nurseries and research departments would suggest that, to bring a new plant variety into routine use, trial and error methods over 18 months were required to gain significant growth and established cultures. Difficult to measure

The cultures that grew were difficult to measure, especially the cauliflower propagation and the carrot callus culture. Any measurement required the removal of the plant material from the culture tube and consequently an increased risk 94

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of contamination. Because the growth occurred over months, it was also a problem to arrange appropriate measuring intervals. Limited student skills

Many of the contamination problems were down to the lack of skills shown by the students. Despite clear demonstrations, students are slower, leave the tissue and tubes exposed for longer, and fail to perform the aseptic technique adequately. Commercial micropropagation nurseries take three months to train up their laboratory staff to use the same aseptic techniques efficiently; it is therefore perhaps unrealistic to expect school students to perform to the required standard in one or two practical sessions. Lack of facilities

Many schools lack the ideal equipment for plant tissue culture, such as transfer chambers and sterile culture tubes and bottles.

Proposed new student protocol Our research has tried to address these issues and produce a method that is: l fast growing; l measurable; l reliable, with a high success rate and a low

rate of contamination problems; l easy for students to learn adequate skills; l possible to set up on a normal laboratory bench without bacterial or fungal contamination; l visually exciting to students. Preparing the medium

The medium was prepared using a modified method developed at the Micropropagation Unit, Royal Botanic Gardens, Kew, and available on the Science and Plants for Schools (SAPS) website. This recipe, originally designed for cauliflower tissue culture, has several advantages. It does not require an autoclave because the sterilising effect of the Milton solution (active chemical dichloroisocyanurate) remains in the medium to provide continuing protection against microbial contamination infection during incubation of the cultures. In this method, the chemicals are dissolved and then the agar is melted in a microwave before the addition of the Milton solution. Because some tenacious fungi continued to cause problems, the method was modified with the addition of 1% Nipagin (methyl p-hydroxybenzoate, also known as

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methylparaben) to inhibit their growth. CLEAPSS and other safety data sheets suggest Nipagin poses no significant hazard, but may be an irritant to skin and eyes. It is recommended that technicians use eye protection when preparing the medium from powder, and that students avoid skin contact with the prepared medium. Suitable containers

Small jars or tubes with screw lids are most suitable. They are easy to place tissue into and they retain moisture for long periods of time. Most school laboratory suppliers have a suitable container in their catalogues. We used 70 cm3 clear plastic containers with screw-on lids (catalogue number BO 03091) from Timstar Laboratory Supplies, Crewe, Cheshire. Purpose-designed tissue culture jars with vented lids can be bought but good growth has been achieved with unvented jars. Containers could be bought ready-sterilised or glass jars could be autoclaved (plastic containers can be microwaved for 30 seconds). The jars need to be filled to a depth of 1 cm with the growth medium. If the jar is unvented the agar will not dry out within the time required for this experiment. We have not seen any adverse effects on growth from the inevitable changes to oxygen and carbon dioxide concentrations over time, although this is something that future experiments need to consider. Petri dishes could be used but they are not ideal because they dry out quickly and need careful sealing, such as with electrical insulating tape. Plant tissue

Garlic meristematic stem disc has proven to be both reliable and fast growing. This follows the work of Ayabe and Sumi (1998; 2001), who produced a technique called the ‘stem-disc dome culture method’ for commercial production of virus-free cultivars. Older and slightly drier garlic bulbs are used in preference to fresh ones. Ayabe and Sumi found that pretreatment by storing the garlic bulbs in a refrigerator at 4 °C for approximately eight weeks enhanced growth and especially shoot development. We have found that even one week of cold storage in a refrigerator can be quite critical: batches of garlic cloves bought fresh from the supermarket showed a low percentage of growth, but the same batch after storage for a week all showed growth after just one day. Zheng et al. (2002) found that many cultivars responded in similar ways to a range

of culture techniques and we have experienced similar success from a wide range of suppliers. The dry scales were removed from a single clove of garlic and a 2 mm thick slice was cut from the basal part of the clove which is the stem disc. The upper part of the clove was discarded. The 2 mm thickness is quite critical as the first millimetre may only consist of dried root tissue from the previous year’s growth but the second millimetre contains the meristematic cells. The cloves within the garlic bulb have each developed from an axillary bud and therefore each represents a condensed shoot. Garlic is a monocotyledonous species and therefore the basal stem disc is meristematic tissue waiting to develop in appropriate conditions. The cells on the basal surface are likely to develop into root tissue and those on the apical surface into shoot tissue. Callus will develop if a suitable growth regulator regimen is supplied. The stem disc was cut in half, sterilised and finally placed on the growth medium. One half-disc was placed with the basal surface down and this tended to encourage shoot and callus growth, which may be a function of the kinetin in the medium stimulating the shoot and inhibiting root growth. The other disc was placed upside down and this showed good root growth. Placing the stem disc halves in this way will therefore demonstrate the full regeneration potential of the tissue. Sterilisation and aseptic technique

The practical was carried out on normal open laboratory benches that had been wiped down with a household antibacterial cleaner. Aseptic technique was used, with scalpel and forceps dipped into a beaker of 70% ethanol to sterilise them before flaming and use. Material was cut inside sterile plastic Petri dishes and lids opened as little as possible to reduce contamination from airborne microbes. The slices of stem disc were sterilised by immersion in 70% ethanol for 2 minutes and then transferred aseptically to a Universal bottle of sterile distilled water containing 1% Nipagin and agitated for 1 minute. After this initial wash to remove the ethanol, the tissue was transferred aseptically to a second identical Universal bottle of sterile distilled water plus Nipagin for a further wash for at least 3 minutes. The tissue was then removed to a sterile Petri dish, ready for placement in the culture jar. SSR June 2012, 93(345)

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Plant tissue culture using stem disc meristem micropropagation of garlic Placement in culture jar

Two pieces of stem disc were placed in each culture jar, one with the basal side upwards and the other with it downwards. The jars were placed in a growth chamber with fluorescent tube lighting providing 10 000 lux, and a photoperiod of 16 hours. The lights provided some heating such that the temperature was maintained at approximately 20 °C. We have achieved good growth on a laboratory windowsill with less light, but night-time temperatures below 10 °C are likely to affect growth adversely. Trying to find the optimum growth conditions is a suitable area for further research but we have found that it is not critical to the success of the practical. Measurement of growth

Our experience suggests that in a class set of cultures there will be both root and shoot growth. For quantitative results, the number of roots and shoots can be counted, and their total length could be estimated since the culture jar must remain closed to maintain sterile conditions. This could

be done from photographs. Growth is obvious from day 3 and should give good results after 10 days (as shown in Figure 1). After 7 days, some cultures could show signs of callus. Development can continue for at least 50 days, producing shoots, roots and bulbils (small bulbs) (Figure 2). Health and safety issues l Wear eye protection when sterilising the discs

of garlic. l The major hazard with carrying out the aseptic technique is the risk of an alcohol fire. The ethanol must be kept well away, at least 1 metre, from the Bunsen flame at all times. Keeping the ethanol in a covered container except when briefly dipping forceps or scalpel into it will reduce the risk. l A sharp scalpel is needed to cut the stem disc and students should be alerted to the need to cut away from hands. l Technicians should be aware that dissolving the Milton tablet, and adding the solution to the medium, generates chlorine, so this must be done in a well-ventilated area.

Figure 1  Stem disc growth after 10 days

Figure 2  Shoot, bulbil and callus development after 25, 30 and 50 days 96

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Student protocol Micropropagation of garlic (Allium sativum L.) using stem disc tissue derived from cloves Garlic is an important crop plant that originated in Central Asia but is now used worldwide for both culinary and medicinal purposes. Most varieties of garlic are sterile and cannot be reproduced by seed. Therefore it is propagated vegetatively by growing individual cloves from the bulb separately. This method has the disadvantages of increasing numbers slowly and passing on viral diseases. New methods, including tissue culture and micropropagation, have been used to solve this problem. In commercial systems, the garlic tissue is cultured to form callus tissue. Callus is a mass of mostly undifferentiated cells that can be grown in laboratory media. This is subdivided into many fragments, each of which can grow back into a new plant using a culture medium containing appropriate levels of plant hormones. The method below is used to establish the initial cultures and has been found capable of producing stem, root and callus tissue from the pieces of meristem cut out of the clove. Materials l cloves of garlic that have been stored at low temperature (4–6 °C) for 2 weeks; l culture jar containing sterile growth medium (Murashige and Skoog basal medium supplemented with sucrose and kinetin); l two sterile plastic Petri dishes; l 100 cm3 beaker containing 70% ethanol for aseptic technique; l sharp scalpel and blunt forceps; l two Universal bottles of sterile distilled water containing 1% Nipagin antifungal chemical; l one specimen tube containing 70% ethanol; l waterproof marker pen. Method 1. Remove all papery scales from the surface of the clove, and, if dry scaly material covers the meristem, break this away leaving firm white tissue. 2. Sterilise a sharp scalpel and cut a 2 mm slice off the base of the clove. This is the stem disc

l Any contaminated cultures should be

autoclaved unopened before disposal. A pressure cooker operating at 15 psi could be used for this purpose. Many modern pressure cookers do not

that includes all the meristematic tissue. 3. Place the stem disc into one of the sterile Petri dishes and cut it into equal halves. 4. Place both pieces of tissue into the specimen tube containing 70% ethanol for 2 minutes. Shake the tube to ensure the surface is fully sterilised. Note: Keep the beaker of ethanol covered and well away from the Bunsen burner. 5. From now on use careful aseptic technique to ensure the tissue remains free of microbial contamination. 6. Transfer the pieces aseptically into the first Universal bottle of sterile distilled water and Nipagin and leave for 1 minute. Shake the bottle to ensure all ethanol is washed off the garlic pieces. 7. Transfer the pieces aseptically into the second Universal bottle of sterile distilled water and Nipagin and leave for at least 3 minutes. Remove the garlic stem disc pieces with a sterile pair of blunt forceps and place inside the second sterile Petri dish. Ensure the Petri dish lid is replaced to prevent contamination from airborne microbes. 8. Aseptically place one piece of stem disc onto the surface of the medium in the culture jar with the stem side down. Place the second piece in the same jar but in the reverse orientation; i.e. with the basal root side down. Ensure the lid of the jar is raised as little as possible, in line with good aseptic technique. 9. Label the lid of the jar: NAME, DATE, GARLIC, 2 min ethanol. 10. On the body of the jar draw a small reference line so that any repeated photographs can be lined up correctly. 11. Place the completed jars in the growth chamber (10‌ 000 lux, 16 hour photoperiod, 20 °C). 12. Observe and record growth changes every 2 days for 3 weeks.

have interchangeable weights but they all operate at a similar pressure of 15 psi, which is equivalent to 2 atmospheres of internal pressure. SSR June 2012, 93(345)

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Conclusion

Acknowledgements

The key aim of investigating tissue culture methods and micropropagation in schools is for students to observe development of callus tissue and whole new plants. As with all practicals involving plant tissue culture, uncontaminated rapid growth is not guaranteed, but experience has shown that starting with garlic stem discs and taking the precautions explained in this article significantly improves the chances that students will achieve a good percentage of growth. The growth is so vigorous that some contamination by fungi will not significantly interfere with the results in the first 10 days.

This work was supported by a Partnership Grant from the Royal Society. Cheadle Hulme School provided a sabbatical half term to allow the author (GP) to start this work. Our thanks to Helen Bradley and Grace Barker of Cheadle Hulme School for running initial trials, Barbara Wright of Micropropagation Services, East Leake, Loughborough, for discussions on commercial applications, and Margaret Ramsey of Kew Gardens for a tour of facilities and discussion on possible lines of research.

References Ayabe, M. and Sumi, S. (1998) Establishment of a novel tissue culture method, stem-disc culture, and its practical application to micropropagation of garlic (Allium sativum L.). Plant Cell Reports, 17, 773–779. Ayabe, M. and Sumi, S. (2001) A novel and efficient tissue culture method – “stem-disc dome culture” – for producing virus free garlic (Allium sativum L.). Plant Cell Reports, 20, 503–507. Fuller, M. P. and Pizzey, T. (2001) Teaching fast and reliable tissue culture using PPM and brassicas. ISHS Acta Horticulturae, 560, 567–570. Storr, T. (1985) Experimenting with Industry. 13. Plant Tissue Culture. Hatfield: Association for Science Education. Available at: www.docstoc.com/

docs/56346770/Experimenting-with-Industry-PlantTissue-Culture. Zheng, S., Henken, B., Krens, F. and Kik, C. (2003) The development of an efficient cultivar-independent plant regeneration system from callus derived from both apical and non-apical root segments of garlic (Allium sativum L.). In Vitro Cellular & Developmental Biology – Plant, 39, 288–292. Website Science and Plants for Schools (SAPS) – Tissue Culture and Micropropagation – Cloning Cauliflowers: www. saps.org.uk/secondary/teaching-resources/706.

Gerry Peat is the head of biology at Cheadle Hulme School, Cheshire. Email: [email protected] Meriel Jones is a senior lecturer in the School of Biological Sciences at the University of Liverpool. Email: [email protected]

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